The vectorization of diagnostic and especially therapeutic tools is a very important stake. Indeed, to bring, thanks to a “vehicle”, therapeutic objects (medicines, physical agents) specifically at the level of the therapeutic targets (typically tumour cells) can allow increasing both the concentration and efficiency of these therapies, while decreasing their side-effects resulting from a nonspecific distribution. So, the use of a highly efficient but very toxic substance can be envisaged with a system of effective targeting. In bioimaging, targeting allows refining the diagnosis by improving the sensibility and especially the specificity for an earlier diagnosis. Qualitative and quantitative study of the internalization in the therapeutic target could previously be done by imaging with the same nanoprobe (organic molecule with a nanometric size in contrast to nanoparticles that are inorganic molecules with a nanometric size) allowing adapting the level of the therapeutically injected radioactivity.
But this objective, pursued by numerous teams in the field of therapy, meets important obstacles related in particular to the non optimal specificity of the vector for the target which ends in a too low ratio [therapeutic object in tissue to treat]/[therapeutic object in healthy tissue]. Such problem is connected to the fact that, except the central nervous system, there are no membrane, enzymatic or other structures which are specific of a single type of cell; in other words, there are no targets totally specific of a given cellular population, but simply an over-expression of these targets. Indeed, the tumor cells only differ from normal cells by an over-expression of cellular “markers”. Thus, the targeting of specific cells by a single marker often comes along with a bad targeting specificity and then large side-effects.
Therefore, a dendritic approach to in vivo efficient targeting seems promising as it combines several advantages such as:                increasing sharply the binding ratio of the nanoprobe on the target tissue by increasing the number of biological effectors within a same nanoprobe;        allowing a multi-modal imaging (magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT)) through complexation of diverse metallic ions;        having favorable biodistribution properties.        
Indeed, elimination of the non targeted complexes is essential and can be done by several routes, in particular by renal route or hepatic and biliary way.
Moreover, the dendritic globular shape could improve the coupling stability with the biological effectors as well as the thermodynamic and kinetic stability of the metallic complex.
The use of dendrimers or dendritic compounds for biomedical applications is a flourishing area of research, mainly because of their precisely defined structure and high tunability, leading to biocompatible, polyfunctional and water-soluble systems (S. E. Stiriba, H. Frey and R. Haag, Angew. Chem. Int. Ed., 2002, 41, 1329-1334; M. J. Cloninger, Curr. Opin. Chem. Biol., 2002, 6, 742-748; R. Duncan and L. Izzo, Adv. Drug Delivery Rev., 2005, 57, 2215-2237; C. C. Lee, J. A. MacKay, J. M. J. Fréchet and F. C. Szoka, Nat. Biotechnol., 2005, 23, 1517-1526; O. Rolland, C-O. Turrin, A-M. Caminade, J-P. Majoral, New J. Chem., 2009, 33, 1809-1824).
Dendritic Contrast Agents and Radiopharmaceuticals
a) Gadolinium-Based Contrast Agents
In recent years, a number of research groups have explored the use of dendrimers as a new class of macromolecular (MRI) contrast agents. The efficiency of MRI contrast agents is often expressed in terms of their longitudinal relaxivity (r1/mM−1·s−1), i.e. their ability to shorten the longitudinal relaxation time of protons of water molecules (T1/s).
In seminal work, Wiener et al. (E. C. Wiener, M. W. Brechbiel, H. Brothers, R. L. Magin, O. A. Gansow, D. A. Tomalia and P. C. Lauterbur, Magn. Reson. Med., 1994, 31, 1) reported the synthesis of different generations of Gd(III)DTPA-based PAMAM dendrimers. Their sixth generation dendritic MRI contrast agent (MW=139 kg·mol-1) displayed an r1 of 34 mM−1·s−1 (0.6 T, 20° C.), which was six times higher than the r1 of Gd(III)DTPA (MW=0.55 kg·mol-1, r1=5.4 mM−1·s−1). This strong increase in r1 was ascribed to the lower molecular tumbling rate of the Gd(III)DTPA complex at the periphery of the dendrimer, as evidenced from the increase in the rotational correlation times (E. C. Wiener, F. P. Auteri, J. W. Chen, M. W. Brechbiel, O. A. Gansow, D. S. Schneider, R. L. Belford, R. B. Clarkson and P. C. Lauterbur, J. Am. Chem. Soc., 1996, 118, 7774). Interestingly, no increase in r1 value was observed for flexible macromolecular polymers of comparable molecular weight (V. S. Vexler, O. Clement, H. Schmitt-Willich and R. C. Brasch, J. Magn. Reson. Imaging, 1994, 4, 381; T. S. Desser, D. L. Rubin, H. H. Muller, F. Qing, S. Khodor, G. Zanazzi, S. W. Young, D. L. Ladd, J. A. Wellons and K. E. Kellar, J. Magn. Reson. Imaging, 1994, 4, 467) implying that segmental motion dominates the rotational correlation time. Bryant et al. investigated the relationship between r1 and the molecular weight of the dendritic MRI contrast agent using different generations of Gd(III)DOTA-based PAMAM dendrimers (L. H. Bryant, Jr, M. W. Brechbiel, C. Wu, J. W. Butte, V. Herynek and J. A. Frank, J. Magn. Reson. Imaging, 1999, 9, 348). In that case, a plateau value for r1 of 36 mM−1·s−1 (0.47 T, 20° C.) was reached for the seventh generation of Gd(III)DOTA-based dendrimer (MW=375 kg·mol−1). Moreover, it was demonstrated that r1 of the seventh generation dendrimer increases with increasing temperature, indicating that slow water exchange limits the relaxivity (E. Toth, D. Pubanz, S. Vauthey, L. Helm and A. E. Merbach, Chem. Eur. J., 1996, 2, 1607). Rudovsky et al. studied the effect on r1 of the ionic interactions between negatively charged Gd(III)-based PAMAM dendrimers and positively-charged poly(aminoacids) (J. Rudovsky, P. Hermann, M. Botta, S. Aime and I. Lukes, Chem. Commun., 2005, 2390). Titration experiments on the second generation dendritic contrast agent with poly(arginine) showed an increase in r1 from 20 to 28 mM−1·s−1 (0.47 T, 20° C.). This effect was attributed to a decrease in the mobility of the Gd(III) complex, induced by interactions between the anionic dendrimer and the cationic poly(arginine). A series of Gd(III)DTPA-functionalized PPI dendrimers was reported by Kobayashi et al. (H. Kobayashi, S. Kawamoto, S.-K. Jo, H. L. Bryant, Jr, M. W. Brechbiel, Jr and R. A. Star, Bioconjugate Chem., 2003, 14, 388). The authors demonstrated that r1 almost linearly increased with the molecular weight of the dendrimer without reaching a plateau value, eventually resulting in a r1 value of 29 mM−1·s−1 (1.5 T, 20° C.) for the fifth generation of dendritic contrast agent. Later on, E. W. Meijer et al. reported a novel series of Gd(III)DTPA-based PPI dendrimers utilizing a different linker between the Gd(III) complex and the dendrimer. (S. Langereis, Q. G. de Lussanet, M. H. P. van Genderen, W. H. Backes and E. W. Meijer, Macromolecules, 2004, 37, 3084) Also, for these dendrimers, a significant increase in r1, though not as pronounced as for the dendritic MRI contrast agents reported by Kobayashi et al., was observed, while molecular weights of both systems were comparable (fifth generation: r1=20 mM−1·s−1, 1.5 T and 20° C.). Researchers at Schering AG (Berlin, Germany) have developed a lysine-based class of dendritic contrast agents: Gadomer-17® (r1=15.2 mM−1·s−1, 1.5 T and 37° C.). (C. Fink, F. Kiessling, M. Bock, M. P. Lichy, B. Misselwitz, P. Peschke, N. E. Fusenig, R. Grobholz and S. Delorme, J. Magn. Reson. Imaging, 2003, 18, 59; G. M. Nicolle, E. Toth, H. Schmitt-Willich, B. Raduchel and A. E. Merbach, Chem. Eur. J., 2002, 8, 1040; G. Adam, J. Neuerburg, E. Spuntrup, A. Muhler, K. Scherer and R. W. Gunther, J. Magn. Reson. Imaging, 1994, 4, 462; G. Adam, J. Neuerburg, E. Spuntrup, A. Muhler, K. Scherer and R. W. Gunther, Magn. Reson. Med., 1994, 32, 622; H. C. Schwickert, T. P. Roberts, A. Muhler, M. Stiskal, F. Demsar and R. C. Brasch, Eur. J. Radiol., 1995, 20, 144; H. C. Roberts, M. Saeed, T. P. Roberts, A. Muhler, D. M. Shames, J. S. Mann, M. Stiskal, F. Demsar and R. C. Brasch, J. Magn. Reson. Imaging, 1997, 7, 331). These macromolecular MRI contrast agents were synthesized from a trimesoyltriamide central core, to which 18 lysine amino acid residues were introduced.
In all these examples, dendrimers have shown to be suitable synthetic scaffolds for the incorporation of multiple Gd(III) moieties, leading to an improved sensitivity for MRI in terms of r1. These conclusions are based on measurements at current magnetic fields of 0.5-1.5 T. However, at high magnetic fields of 10 T, the r1 values of dendritic contrast agents are substantially lower, not exceeding the r1 values of low molecular weight Gd(III)-based complexes. Dendrimers also improve the protection of the gadolinium and its stability and thus decrease the toxicity risks.
The dendritic MRI contrast agents are excellent blood pool agents. However, these structures lack the specificity required for molecular MRI (D. Artemov, J. Cell. Biochem., 2003, 90, 518). The development of target-specific MRI contrast agents, directed to defined molecular markers, could dramatically improve the targeting and imaging of a specific disease, due to the accumulation of MRI contrast agent at the region of interest.
b) Dendritic Radiopharmaceuticals
The use of dendrimers for the complexation of 99mTc was scarcely reported in the literature so far: in 2001, F. Vögtle et al. (H. Stephan, H. Spies, B. Johannsen, K. Gloe, M. Gorka, F. Vögtle, Eur. J. Inorg. Chem., 2001, 2957-2963) reported host-guest properties of multi-crown dendrimers of four different generations towards sodium pertechnetate. Extraction studies performed showed that the guest molecules are mainly bound in the interior of the polyamine squeleton. The same year, H. Mukhtar and coll. (M. Subbarayan, S. J. Shetty, T. S. Srivastava, O. P. D. Noronha, A. M. Samuel, H. Mukhtar, Biochem. and Biophys. Res. Commun., 2001, 281, 32-36) reported the synthesis and in vivo distribution of water-soluble 99mTc-labeled dendritic porphyrins for tumor imaging and diagnosis: these dendritic systems were administered to C6-glioma tumor bearing Wistar rats and scinti-imaging studies showed their potential for early stage tumor detection. Finally, A. Adronov, J. F. Valliant et al (M. C. Parrott, S. R. Benhabbour, C. Saab, J. A. Lemon, S. Parker, J. F. Valliant, A. Adronov, J. Am. Chem. Soc., 2009, 131, 2906-2916) published very recently a paper dealing with the use of high-generation polyester dendrimers to complex 99mTc and their use for SPECT imaging: it was found that all three dendrimer generation (G5 to G7) were rapidly and efficiently removed from the bloodstream via the kidneys and excreted through the bladder within 15 min post injection. The SPECT-CT data were corroborated with a quantitative biodistribution study involving ex vivo harvesting of various organs and determining the radioactivity within the organs as a function of time.
The international application WO2008/043911, relates to chelated dendritic complexes and their applications biomedical imaging; such complexes have the following formula:[[MC]-En-[D]m-X1p1X2p2X3P3X4P4]Z−zB+wherein:    M is a magnetic cation, in particular chosen among Gd3+, Mn2+ and 99mTc3+,    C is a chelating agent of the magnetic marker M,    [MC] is a chelate of the magnetic marker M,    E is a spacer,    n=0 or 1,    [D] is a dendritic structure having a core comprising at least one group derived from benzyl alcohol or a benzylamine, the benzyl cycle of which is substituted in positions 3, 4, 5 by dendrites composed of polyethyleneglycol pattern,    m is an integer being equal to 1 or 2 or 4,    Xi is a group increasing the complex lipophily, such as a tert-butyl group (tBu),    p1 is an integer from 0 to 12,    X2 is a group increasing the complex specificity for a particular organ, preferably for the brain, such as L-dopamine,    p2 is an integer being equal to 0, 1, 2, or 4,    X3 un group having a therapeutic activity, preferably for neurodegeneratives diseases such as Alzheimer disease, Parkinson disease and multiple sclerosis,    p3 is an integer equal to 0, 1, 2, or 4,    X4 is a CH3 group,    p4 is an integer from 0 to 12    p1+p2+p3+p4=3 when m=1 or p1+p2+p3+p4=6 when m=2 or p1+p2+p3+p4=12 when m=4,    B counter ion, preferably Na+ or K+,    z is an integer equal to 0, 1, 2, 3 or 4.
Nevertheless, the application WO2008/043911 is very confusing for a man skilled in the art because:                on one hand preferred compounds are complexes of the formula defined above wherein dendrites of each structure [D] are functionalized with L-Dopamine but it is well known for a man skilled in the art that dopamine does not cross the blood brain barrier and further the specification discloses a dendrite [D] that is not functionalized with L-Dopamine but with a 3,4 OH phenylglycine.        on the other hand, example 2 discloses the synthesis of compound of formula III-1 to 111-3 but said synthesis could not be achieved as the compound disclosed before the reaction with a metallic moiety could not lead at all said formulas.        
Thus in view of this international application, a man skilled in the art does not know the exact functionalities to be introduced in the general formula to give an imaging agent with brain specificity.
An article published in New Journal of chemistry (2010), 34, 267-275, disclosed compounds having the following structures 1 and 2:
as imaging agents. No functionalization of the oligoethylene pattern group is described, but it is concluded that grafting L-Dopamine to the dendron periphery will allow the elaboration of brain-targeting radiopharmaceuticals.
Thus the teaching of this document is as confusing as the one of the international application WO2008/043911.